Targeted Protein Degradation by Chimeric Compounds using Hydrophobic E3 Ligands and Adamantane Moiety.

Targeted protein degradation using small chimeric molecules, such as proteolysis-targeting chimeras (PROTACs) and specific and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs), is a promising technology in drug discovery. We recently developed a novel class of chimeric compounds that recruit the aryl hydrocarbon receptor (AhR) E3 ligase complex and induce the AhR-dependent degradation of target proteins. However, these chimeras contain a hydrophobic AhR E3 ligand, and thus, degrade target proteins even in cells that do not express AhR. In this study, we synthesized new compounds in which the AhR ligands were replaced with a hydrophobic adamantane moiety to investigate the mechanisms of AhR-independent degradation. Our results showed that the compounds, 2, 3, and 16 induced significant degradation of some target proteins in cells that do not express AhR, similar to the chimeras containing AhR ligands. However, in cells expressing AhR, 2, 3, and 16 did not induce the degradation of other target proteins, in contrast with their response to chimeras containing AhR ligands. Overall, it was suggested that target proteins susceptible to the hydrophobic tagging system are degraded by chimeras containing hydrophobic AhR ligands even without AhR.

Development of Small Molecule Chimeras That Recruit AhR E3 Ligase to Target Proteins.

Targeted protein degradation using chimeric small molecules such as proteolysis-targeting chimeras (PROTACs) and specific and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) is an emerging modality in drug discovery. Here, we expand the repertoire of E3 ligases capable of ubiquitylating target proteins using this system. By incorporating ß-naphthoflavone (ß-NF) as a ligand, we developed a novel class of chimeric molecules that recruit the arylhydrocarbon receptor (AhR) E3 ligase complex. ß-NF-ATRA, a chimeric degrader directed against cellular retinoic acid binding proteins (CRABPs), induced the AhR-dependent degradation of CRABP-1 and CRABP-2 via the ubiquitin-proteasome pathway. A similar compound ITE-ATRA, in which an alternative AhR ligand was used, also degraded CRABP proteins. Finally, we developed a chimeric compound ß-NF-JQ1 that is directed against bromodomain-containing (BRD) proteins using ß-NF as an AhR ligand. ß-NF-JQ1 induced the interaction of AhR and BRD proteins and displayed effective anticancer activity that correlated with protein knockdown activity. These results demonstrate a novel class of chimeric degrader molecules based on the ability to bring a target protein and an AhR E3 ligase into close proximity.

Design and synthesis of novel selective estrogen receptor degradation inducers based on the diphenylheptane skeleton.

Estrogen receptors (ERs) are a family of nuclear receptors (NRs) that regulate physiological effects such as reproduction and bone homeostasis. It has been reported that approximately 70% of human breast cancers are hormone-dependent and ERα-positive. Recently, novel anti-breast cancer drugs based on different mechanisms of action have received significant attention. In this article, we have designed and synthesized a selective ER degradation inducer based on the diphenylheptane skeleton. Western blotting analysis revealed that PBP-NC10 degraded ERα through the ubiquitin-proteasome system. We also performed computational docking analysis to predict the binding mode of PBP-NC10 to ERα.

Tamoxifen and Fulvestrant Hybrids Showed Potency as Selective Estrogen Receptor Down-Regulators.

BACKGROUND: Estrogen receptors (ERs) are an important target for the management of breast cancers. Selective estrogen receptor down-regulators (SERDs) block ER activity, as well as reduce ERα protein levels in cells, and therefore are promising therapeutic agents for the treatment of breast cancers. OBJECTIVE: In order to develop potent SERDs, we prepared tamoxifen and fulvestrant hybrids and evaluated their binding activity and down-regulation of ERα. METHODS: We designed and synthesized tamoxifen derivatives, which had a 4,4,5,5,5- pentafluoropentyl group on the terminal alkyl chain. The oxidation state of the sulfur atom and alkyl length between the sulfur and nitrogen atoms were varied. Western blotting was performed to determine the ability to down-regulate ERα. Binding affinities of synthesized compounds were evaluated by a fluorescence polarization-based competitive binding assay. RESULTS: We successfully prepared nine compounds. Treatment with 11, 14, and 17 effectively reduced ERα protein levels in MCF-7 cells in a concentration-dependent manner. This reduction was inhibited by a proteasome inhibitor. The ability of 14 to down-regulate the ERα protein level was equal to fulvestrant. All compounds showed a largely equal affinity for ERα. CONCLUSION: As indicated by Western blots, the ERα degradation activity was observed only in the series of butyl linker derivatives, namely, 11, 14, and 17. These findings suggest that the specific length of the alkyl chain is an important factor in controlling the down-regulation of ER. These results provide useful information for designing promising SERD candidates.

Synthesis and evaluation of raloxifene derivatives as a selective estrogen receptor down-regulator.

Estrogen receptors (ERs) play a major role in the growth of human breast cancer cells. A selective estrogen receptor down-regulator (SERD) that acts as not only an inhibitor of ligand binding, but also induces the down-regulation of ER, would be useful for the treatment for ER-positive breast cancer. We previously reported that tamoxifen derivatives, which have a long alkyl chain, had the ability to down-regulate ERα. With the aim of expanding range of the currently available SERDs, we designed and synthesized raloxifene derivatives, which had various lengths of the long alkyl chains, and evaluated their SERD activities. All compounds were able to bind ERα, and RC10, which has a decyl group on the amine moiety of raloxifene, was shown to be the most potent compound. Our findings suggest that the ligand core was replaceable, and that the alkyl length was important for controlling SERD activity. Moreover, RC10 showed antagonistic activity and its potency was superior to that of 4,4'-(heptane-4,4-diyl)bis(2-methylphenol) (18), a competitive antagonist of ER without SERD activity. These results provide information that will be useful for the development of promising SERDs candidates.

Synthesis and evaluation of tamoxifen derivatives with a long alkyl side chain as selective estrogen receptor down-regulators.

Estrogen receptors (ERs) play a major role in the growth of human breast cancer cells. An antagonist that acts as not only an inhibitor of ligand binding but also an inducer of the down-regulation of ER would be useful for the treatment for ER-positive breast cancer. We previously reported the design and synthesis of a selective estrogen receptor down-regulator (SERD), (E/Z)-4-(1-{4-[2-(dodecylamino)ethoxy]phenyl}-2-phenylbut-1-en-1-yl)phenol (C12), which is a tamoxifen derivative having a long alkyl chain on the amine moiety. This compound induced degradation of ERα via a proteasome-dependent pathway and showed an antagonistic effect in MCF-7 cells. With the aim of increasing the potency of SERDs, we designed and synthesized various tamoxifen derivatives that have various lengths and terminal groups of the long alkyl side chain. During the course of our investigation, C10F having a 10-fluorodecyl group on the amine moiety of 4-OHT was shown to be the most potent compound among the tamoxifen derivatives. Moreover, computational docking analysis suggested that the long alkyl chain interacted with the hydrophobic region on the surface of the ER, which is a binding site of helix 12 and coactivator. These results provide useful information to develop promising candidates as SERDs.